# US 12,180,472 B2 · Chiral design

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- Number: US 12,180,472 B2
- Document ID: US12180472B2
- Status: Granted
- Kind: B2
- Application number: 17/016,313
- Filed: 2020-09-09
- Published: 2021-04-22
- Granted: 2024-12-31
- Earliest claimed: 2014-01-16
- Assignees: Wave Life Sciences Ltd.
- Applicants: Wave Life Sciences Ltd.
- Inventors: Meena Meena; David Butler; Naoki Iwamoto; Nenad Svrzikapa; Gregory L. Verdine; Ivan Zlatev
- Web page: https://patentgrep.com/patent/US12180472B2

## CPC

- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 2310/31: of the backbone
- C12N 2310/315: Phosphorothioates
- C12N 2320/30: Special therapeutic applications
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to chirally controlled oligonucleotides of select designs, chirally controlled oligonucleotide compositions, and methods of making and using the same. In some embodiments, a provided chirally controlled oligonucleotide composition provides different cleavage patterns of a nucleic acid polymer than a reference oligonucleotide composition. In some embodiments, a provided chirally controlled oligonucleotide composition provides single site cleavage within a complementary sequence of a nucleic acid polymer.

## Claims

### Claim 1

A method for cleaving a nucleic acid polymer, comprising contacting the nucleic acid polymer with a composition, wherein the composition is a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type defined by having:
1) a common base sequence and length;
2) a common pattern of backbone linkages; and
3) a common pattern of backbone chiral centers,
which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length, for oligonucleotides of the particular oligonucleotide type, wherein:
the common base sequence has at least 17 bases;
at least 50% of chiral internucleotidic linkages in each oligonucleotide of the particular oligonucleotide type are Sp; and
the common pattern of backbone chiral centers comprises from 5′ to 3′ Rp(Sp)2.

### Claim 2 (depends on 1)

The method of claim 1, wherein each chiral, modified phosphate linkage of the oligonucleotides of the particular oligonucleotide type independently has the structure of formula I:
wherein:
P* is an asymmetric phosphorus atom and is either Rp or Sp;
W is O, S or Se;
each of X, Y and Z is independently —O—, —S—, —N(-L-R1)—, or L;
L is a covalent bond or an optionally substituted, linear or branched C1-C10 alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C1-C6 alkylene, C1-C6 alkenylene, —C≡C—, —C(R′)2—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)2—, —S(O)2N(R′)—, —N(R′)S(O)2—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;
R1 is halogen, R, or an optionally substituted C1-C50 aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C1-C6 alkylene, C1-C6 alkenylene, —C≡C—, —C(R′)2—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)2—, —S(O)2N(R′)—, —N(R′)S(O)2—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;
each R′ is independently —R, —C(O)R, —CO2R, or —SO2R, or:
two R′ on the same nitrogen are taken together with their intervening atoms to form an optionally substituted heterocyclic or heteroaryl ring, or
two R′ on the same carbon are taken together with their intervening atoms to form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring;
-Cy- is an optionally substituted bivalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, or heterocyclylene;
each R is independently hydrogen, or an optionally substituted group selected from C1-C6 aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl; and
each
independently represents a connection to a nucleoside.

### Claim 3 (depends on 2)

The method of claim 2, wherein W is O, X is —S—, and Y and Z are —O—.

### Claim 4 (depends on 2)

The method of claim 2, wherein X is —S— and -L-R1 is not hydrogen.

### Claim 5 (depends on 1)

The method of claim 1, wherein the pattern of backbone chiral centers comprises (Sp)2Rp(Sp)2.

### Claim 6 (depends on 5)

The method of claim 5, wherein the common base sequence has at least 20 nucleobases.

### Claim 7 (depends on 6)

The method of claim 6, wherein at least 60% of chiral internucleotidic linkages in each oligonucleotide of the particular type are Sp.

### Claim 8 (depends on 5)

The method of claim 5, wherein each of the Sp and Rp in the (Sp)2Rp(Sp)2 is independently the configuration of a phosphorothioate diester linkage.

### Claim 9 (depends on 8)

The method of claim 8, wherein each chiral phosphate linkage of the oligonucleotides of the particular oligonucleotide type is a phosphorothioate diester linkage.

### Claim 10 (depends on 5)

The method of claim 5, wherein the oligonucleotides of the particular oligonucleotide type each comprise one or more phosphate diester linkages.

### Claim 11 (depends on 5)

The method of claim 5, wherein the nucleobases of the oligonucleotides of the particular oligonucleotide type are independently selected from adenine, thymine, cytosine, guanine, uracil and 5-methylcytosine.

### Claim 12 (depends on 5)

The method of claim 5, wherein the oligonucleotides of the particular oligonucleotide type each comprise a modified sugar.

### Claim 13 (depends on 12)

The method of claim 12, wherein the modification of the modified sugar is 2′-OR1, wherein R1 is optionally substituted C1-6 aliphatic.

### Claim 14 (depends on 12)

The method of claim 12, wherein the modification of the modified sugar is 2′-OMe.

### Claim 15 (depends on 12)

The method of claim 12, wherein the modification of the modified sugar is 2′-OCH2CH2OMe.

### Claim 16 (depends on 12)

The method of claim 12, wherein the oligonucleotides of the particular oligonucleotide type each comprise a locked nucleic acid sugar.

### Claim 17 (depends on 12)

The method of claim 12, wherein the modified sugar comprises a bivalent substituent -L-, wherein -L- is —O—CH2— between C2 and C4 of the sugar, wherein the —CH2— is optionally substituted.

### Claim 18 (depends on 12)

The method of claim 12, wherein the modified sugar comprises a bivalent substituent -L-, wherein -L- is —O—CH(Et)- between C2 and C4 of the sugar.

### Claim 19

A method for inhibiting expression or reducing activity of a protein, comprising contacting a nucleic acid polymer encoding the protein with a composition, wherein the composition is a chirally controlled oligonucleotide composition comprising oligonucleotides of a particular oligonucleotide type defined by having:
1) a common base sequence and length;
2) a common pattern of backbone linkages; and
3) a common pattern of backbone chiral centers,
which composition is chirally controlled in that it is enriched, relative to a substantially racemic preparation of oligonucleotides having the same base sequence and length, for oligonucleotides of the particular oligonucleotide type, wherein:
the common base sequence has at least 17 bases;
at least 50% of chiral internucleotidic linkages in each oligonucleotide of the particular oligonucleotide type are Sp; and
the common pattern of backbone chiral centers comprises from 5′ to 3′ Rp(Sp)2; wherein the common base sequence is complementary to that of a nucleic acid encoding the protein.

### Claim 20 (depends on 19)

The method of claim 19, wherein at least 60% of chiral internucleotidic linkages in each oligonucleotide of the particular type are Sp.

### Claim 21 (depends on 20)

The method of claim 20, wherein the pattern of backbone chiral centers comprises (Sp)2Rp(Sp)2.

### Claim 22 (depends on 21)

The method of claim 21, wherein each of the Sp and Rp in the (Sp)2Rp(Sp)2 is independently the configuration of a phosphorothioate diester linkage.

### Claim 23 (depends on 1)

The method of claim 1, wherein each of the Sp and Rp in the Rp(Sp)2 is independently the configuration of a phosphorothioate diester linkage.

### Claim 24 (depends on 19)

The method of claim 19, wherein each of the Sp and Rp in the Rp(Sp)2 is independently the configuration of a phosphorothioate diester linkage.

### Claim 25 (depends on 19)

The method of claim 19, wherein each chiral, modified phosphate linkage of the oligonucleotides of the particular oligonucleotide type independently has the structure of formula I:
wherein:
P* is an asymmetric phosphorus atom and is either Rp or Sp;
W is O, S or Se;
each of X, Y and Z is independently —O—, —S—, —N(-L-R1)—, or L;
L is a covalent bond or an optionally substituted, linear or branched C1-C10 alkylene, wherein one or more methylene units of L are optionally and independently replaced by an optionally substituted C1-C6 alkylene, C1-C6 alkenylene, —C≡C—, —C(R′)2—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)2—, —S(O)2N(R′)—, —N(R′)S(O)2—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;
R1 is halogen, R, or an optionally substituted C1-C50 aliphatic wherein one or more methylene units are optionally and independently replaced by an optionally substituted C1-C6 alkylene, C1-C6 alkenylene, —C≡C—, —C(R′)2—, -Cy-, —O—, —S—, —S—S—, —N(R′)—, —C(O)—, —C(S)—, —C(NR′)—, —C(O)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(O)—, —N(R′)C(O)O—, —OC(O)N(R′)—, —S(O)—, —S(O)2—, —S(O)2N(R′)—, —N(R′)S(O)2—, —SC(O)—, —C(O)S—, —OC(O)—, or —C(O)O—;
each R′ is independently —R, —C(O)R, —CO2R, or —SO2R, or:
two R′ on the same nitrogen are taken together with their intervening atoms to form an optionally substituted heterocyclic or heteroaryl ring, or
two R′ on the same carbon are taken together with their intervening atoms to form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring;
-Cy- is an optionally substituted bivalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, or heterocyclylene;
each R is independently hydrogen, or an optionally substituted group selected from C1-C6 aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl; and
each
independently represents a connection to a nucleoside.

### Claim 26 (depends on 25)

The method of claim 25, wherein W is O, X is —S—, and Y and Z are —O—.

### Claim 27 (depends on 21)

The method of claim 21, wherein the oligonucleotides of the particular oligonucleotide type each comprise a modified sugar, wherein the modification of the modified sugar is 2′—OR1, wherein R1 is optionally substituted C1-6 aliphatic.